Neutrophils facilitate the epicardial regenerative response after zebrafish heart injury

Despite extensive studies on endogenous heart regeneration within the past 20 years, the players involved in initiating early regeneration events are far from clear. Here, we assessed the function of neutrophils, the firstresponder cells to tissue damage, during zebrafish heart regeneration. We detected rapid neutrophil mobilization to the injury site after ventricular amputation, peaking at 1-day post-amputation (dpa) and resolving by 3 dpa. Further analyses indicated neutrophil mobilization coincides with peak epicardial cell proliferation, and recruited neutrophils associated with activated, expanding epicardial cells at 1 dpa. Neutrophil depletion inhibited myocardial regeneration and significantly reduced epicardial cell expansion, proliferation, and activation. To explore the molecular mechanism of neutrophils on the epicardial regenerative response, we performed scRNA-seq analysis of 1 dpa neutrophils and identified enrichment of the FGF and MAPK/ERK signaling pathways. Pharmacological inhibition of FGF signaling indicated its' requirement for epicardial expansion, while neutrophil depletion blocked MAPK/ERK signaling activation in epicardial cells. Ligand-receptor analysis indicated the EGF ligand, hbegfa, is released from neutrophils and synergizes with other FGF and MAPK/ERK factors for induction of epicardial regeneration. Altogether, our studies revealed that neutrophils quickly motivate epicardial cells, which later accumulate at the injury site and contribute to heart regeneration.